Cancer stem cells reside in hypoxic areas and contribute to tumor progression. These cells are particularly difficult to treat because they are inherently resistant to conventional therapy, and their microenvironment reduces the efficacy of radiation and impairs delivery of chemotherapy. Glioblastoma is an incurable primary brain tumor that is characterized by regions of hypoxia. Notch signaling maintains GSCs within the hypoxic niche, but how Notch is dysregulated is unclear. Our preliminary data strongly suggest that hypoxia can regulate membranous Notch levels, thereby regulating receptor availability for ligand binding. We have found that the recently identified hypoxia-regulated protein, Vasorin, is preferentially expressed in GSCs to regulate Notch signaling. Our data suggest that Vasorin regulates GSC self-renewal by inhibiting the lysosomal degradation of Notch1. Thus, we have identified a novel mechanism by which hypoxia directs Notch signaling: regulation of receptor turnover. Our central hypothesis is that Vasorin regulates GSC self-renewal within the hypoxic niche by regulating Notch signaling and that targeting Vasorin may improve GBM therapy. We will test our hypothesis through mechanistic and preclinical studies. In Aim 1, we will determine the role of Vasorin in mediating GSC properties and tumorigenic potential in vitro and in vivo. In Aim 2, we will define the role of Vasorin in regulating Notch signaling to promote GSC self-renewal under hypoxic conditions. In Aim 3, we will assess Vasorin as a prognostic biomarker and provide proof-of-principle that maximizing Notch inhibition by dual targeting of Vasorin and Notch proteolytic processing can improve survival in mouse models of glioblastoma. Importantly, Vasorin does not appear to be expressed in normal embryonic or adult brain. Therefore, targeting Vasorin may have limited normal brain toxicity. These data will reveal an important role for Vasorin in regulating Notch signaling and have significant clinical ramifications. Current therapies using hypoxia modifiers or Notch pathway inhibitors, such as ?-secretase inhibitors, are limited by side effects. If successful, the findings of this study will provide a strong rationale for the development of novel therapeutics against Vasorin.